Vitrification of Human Germinal Vesicle Oocytes: before or after In Vitro Maturation?

Background The use of immature oocytes derived from stimulated cycles could be of great importance, particularly for urgent fertility preservation cases. The current study aimed to determine whether in vitro maturation (IVM) was more successful before or after vitrification of these oocytes. Materials and Methods This prospective study was performed in a private in vitro fertilization (IVF) center. We collected 318 germinal vesicle (GV) oocytes from 104 stimulated oocyte donation cycles. Oocytes were divided into two groups according to whether vitrification was applied at the GV stage (group 1) or in vitro matured to the metaphase II (MII) stage and then vitrified (group 2). In the control group (group 3), oocytes were in vitro matured without vitrification. In all three groups, we assessed survival rate after warming, maturation rate, and MII-spindle/chromosome configurations. The chi-square test was used to compare rates between the three groups. Statistical significance was defined at P<0.05 and we used Bonferroni criterion to assess statistical significance regarding the various pairs of groups. The Statistical Package for the Social Sciences version 17.0 was used to perform statistical analysis. Results There was no significant difference in the survival rate after vitrification and warming of GV (93.5%) and MII oocytes (90.8%). A significantly higher maturation rate occurred when IVM was performed before vitrification (82.9%) compared to after vitrification (51%). There was no significant difference in the incidence of normal spindle/ chromosome configurations among warmed oocytes matured in vitro before (50.0%) or after (41.2%) vitrification. However, a higher incidence of normal spindle/chromosome configurations existed in the in vitro matured oocytes which were not subjected to vitrification (fresh oocytes, 77.9%). Conclusion In stimulated cycles, vitrification of in vitro matured MII oocytes rather than GV oocytes seems to be more efficient. This approach needs to be verified in nonstimulated fertility preservation cases.


Introduction
opened new possibilities and options for oocyte cryoused in most in vitro fertilization (IVF) laboratories as a routine procedure (1-3). The high survival rates that follow warming have made oocyte banking feasible for heterologous use, fertility preservation for social reasons, in cases where women face the dangers of premature ovarian failure, as well as prior to chemooocytes may easily serve as a rescue plan in IVF cycles when the male partner is not able to produce a semen sample at the time of oocyte retrieval. Under the above circumstances, a number of immature oocytes are often retrieved from a stimulated cycle. The majority of collected oocytes from in vitro maturation (IVM) cycles are immature and at the germinal vesicle (GV) stage (7). GV oocytes collected for fertility preservation in women who need an urgent onset of chemo-or radiotherapeutic treatments may be of great value since they can be either matured and cryopreserved or fertilized and cultured in vitro (8,9). In such cases, the question is whether oocytes should be due to the multiple steps of this procedure and different outcomes of each step. Egerszegi et al. (12) in vitro matured metaphase II (MII) pig oocytes. They reported a MII compared to GV oocytes. However, both groups had similar maturation rates; most importantly, the and F-actin integrity. The study concluded that vitin terms of cleavage and blastocyst formation compared to the in vitro matured MII oocytes. On the of immature GV oocytes and in vitro matured MII oocytes concluded that poor maturation and low fertilization rates were major problems associated It is widely accepted that oocytes which fail to mature in vivo under ovarian gonadotropin stimulation and human chorionic gonadotrophin (hCG) trigger are intrinsically abnormal. They present a high incidence of aneuploidies and low developmental competence compared to oocytes that have reached the MII stage at the moment of retrieval (19-22). Additionally, the probability of denuded GV oocytes to mature in vitro cumulus cell communication (23).
In the present study, the recruitment of immature oocytes from stimulated cycles of oocyte donors represented an experimental model that used easily tion of a methodology to rescue immature oocytes of to investigate whether IVM might be more success-rived from stimulated cycles were subjected to IVM terms of survival rate, maturation rate, and the status of in vitro.
We conducted this prospective study from Janureproduction unit. The Institutional Review Board study and we obtained informed consents from all couples that received eggs from their dedicated donors. The IVF Unit computerized database contains all patient characteristics as well as parameters related with stimulation protocol and gamete handling. These data are regularly recorded and revalidated on a monthly basis by specialized personnel in order to maintain data reliability. GV oocytes were obtained from controlled ovarian hyperstimulation oocyte do-2 years. We randomly allocated the GV oocytes into and matured in vitro immature oocytes underwent IVM and those that were placed in maturation medium and served as the by immunostaining in all three groups. MII oocytes that presented with partial or complete disorganization of the spindle poles or complete absence of the meiotic spindle were characterized as abnormal.
mg, Organon) with 225 IU/day of recombinant follicle stimulation hormone (FSH, Puregon, Organon) that started on day 2 of the cycle for ovarian stimulation (19). The daily dose of recombinant FSH was adjusted according to the donor's ovarian response based on serum estradiol concentrations and the Pregnyl, Organon) was administered when three or more follicles >17 mm in mean diameter were present on ultrasound and a serum estradiol concentra-tion received an additional dose of GnRH-antagonist on the day before hCG administration (24). The retrieved oocytes were incubated for 2 hours in equilibrated Quinn's Advantage Fertilization (HTF, Sage, Copper Surgical, USA) supplemented with 5% human serum albumin (HSA, Sage, Copper Surgical, 2 . Afterwards, they were (Sage, Copper Surgical, USA).

In vitro
We placed the GV oocytes in maturation medium (Sage, Copper Surgical, USA) supplemented with 75 mIU/ml FSH, 75 mIU/ml luteinizing hormone Sage, Copper Surgical, USA) for 24-48 hours after oocyte denudation. Next, we determined oocyte The total time of incubation after oocyte retrieval until the second control, or 62 and 86 hours post hCG administration, respectively.
We used a closed carrier system (VitriSafe, Vitchemical substances were purchased by Sigma-Aldrich unless otherwise mentioned. A mixture of dimethyl sulphoxide (DMSO) and ethylene glycol (EG) was used in dilutions of 1.25%/1. Primary outcomes included overall maturation rate after warming. The maturation rate 62 hours after hCG administration was considered a secondary outcome. oocytes that survived after warming to the overall MII oocytes to the number of oocytes that underwent IVM. The maturation rate after 62 hours was the overall number of oocytes that underwent IVM. spindle after IVM to the total number of oocytes that survived in each group.
The categorical variables were expressed as percentages. We used the chi-square test to compare rates various group pairs (group 1 versus group 2, group 1 versus group 3, group 2 versus group 3). Odds raestimated for comparisons between pairs of groups.
Results the survival rate after warming between groups 1 ence between the two groups. The survival rate was Maturation group 1. The overall maturation rate was 82.9% for ---

Discussion
Results from previous studies indicate that vitri-sible option (29-31). Various proposed protocols the processess of nuclear and cytoplasmic oocyte maturation and maintain further, normal embryo development (32, 33). The immature oocytes assessed in the present study have been retrieved low developmental potential (34). However, they could still be used as a research model to investi-man oocytes.
Our results, being in accordance with recent ference in the survival rate of immature oocytes tion protocol. However, the maturation process appeared to be compromised when intercepted by the rate (51%) compared to oocytes that were in vitro cation needed a longer culture time in maturation media in order to reach the MII stage compared study by Wang et al. (36). Data from both studies did not verify the hypothesis and general belief that the most appropriate time to vitrify oocytes would be at the GV rather than MII stage (12, 37). Although, at the GV stage, the chromatin is diffused in the diplotene state of prophase I and well protected by the nuclear membrane, which suggests that these oocytes are less vulnerable to the risk of chromosome missegregations. This study ly compromised the progress of oocyte maturation and rate of fertilization (6, 36).  28) demonstrated that fol-man embryos increased. These abnormalities were mainly due to dehydration and mechanical stress sustained by the cells and not cryoprotectant toxicity. However, DNA repair mechanisms might be activated to rescue both oocytes and embryos fol-Despite the fact that embryos derived from in vitro developmental competence, they possibly provide a person's unique opportunity to fecundity. This attribute has tremendously increased their value and the need to be processed in the best possible way. However, due to the lack of experience on utilization of embryos produced from such oocytes, extensive counseling and prenatal genetic testing should be considered.

Conclusion
The results of the present study indicated that in vitro matured or GV oocytes was equally successful. However, the maturation process of the GV applied on in vitro matured MII oocytes. The pre-ther studies, preferably on non-stimulated cycles where most oocytes are collected at the GV stage.